Stabilizing a Femur Osteotomy with a Plate Fixation in Ambystoma mexicanum

Abstract

The axolotl (Ambystoma mexicanum) is a promising model organism for regenerative medicine due to its remarkable ability to regenerate lost or damaged organs, including limbs, brain, heart, tail, and others. Studies on axolotl shed light on cellular and molecular pathways ruling progenitor activation and tissue restoration after injury. This knowledge can be applied to facilitate the healing of regeneration-incompetent injuries, such as bone non-union. In the current protocol, the femur osteotomy stabilization using an internal plate fixation system is described. The procedure was adapted for use in aquatic animals (axolotl, Ambystoma mexicanum). ≥20 cm snout-to-tail tip axolotls with fully ossified, mouse-size comparable femurs were used, and special attention was paid to the plate positioning and fixation, as well as to the postoperative care. This surgical technique allows for standardized and stabilized bone fixation and could be useful for direct comparison to axolotl limb regeneration and analogous studies of bone healing across amphibians and mammals.

Introduction

The axolotl (Ambystoma mexicanum) is an important model for organ regeneration, including the tail, spinal cord, brain, heart, gills, and limbs^1,2,3,4,5. Detailed studies of axolotl limb regeneration uncovered mechanisms of cell dedifferentiation and the formation of a stem cell pool, blastema, at the amputation site. Due to the ability of the blastema cells to reconstruct all missing limb parts, including a patterned skeleton^6,7, the axolotl appears to be an attractive model organism for bone healing studies. Recently, several studies focused more on bone biology in axolotls, describing skeletal morphology, cellular composition, and ossification dynamics.

It was found in mammals that the bone healing process in long bones occurs via endochondral ossification and consists of several stages: hematoma, granulation tissue, and soft callus formation, callus ossification into hard callus and woven bone, and bone remodeling⁸. A recent study has shown that similar stages can be observed in axolotl bone healing⁹.

Until now, axolotl fractures were studied in a non-stabilized system, whereby bone is simply cut with iridectomy scissors. The large fractures were created in the zeugopod, where osteotomy is performed on one of the bones, whereas the other serves as support^10,11. In contrast, fractures are routinely studied in mammals, including rats and mice, using reliable fixation systems, such as intramedullary pin and bone-aligning plates, to control fracture size and ensure bone alignment.

Thus, the method aims to ensure stabilized and uniform fixation of the axolotl femur prior to osteotomy. In order to make axolotl studies more comparable to mammals, including mice and humans, intramedullary pin¹², external plate fixator^13,14, and internal bone aligning plate^15,16,17fixation were considered. The latter was shown to ensure proper bone fixation and allow for creating a gap of a certain size by using one or two cuts with a Gigly saw of a specific diameter. As axolotls represent the aquatic larvae of Ambystoma mexicanum, the external fixator might have caused post-surgical complications due to the open wound and contact with water. As axolotls do not develop secondary ossification centers even until very late in their development (20 years old¹⁸), and thus the standard intramedullary nail used in mice might not be prevented from puncturing the epiphyses, a decision was made to apply an internal plate fixation method to large axolotls. In large axolotls, the femur size and degree of ossification resemble that of an adult mouse, thus allowing for mid-diaphyseal osteotomy with titanium plate fixation¹.

The fracture gap size largely determines the healing dynamics and outcome. For example, in a mouse, a 0.25 mm stabilized fractures heal mostly through intramembranous ossification due to their small size and rigid stabilization; a 0.7 mm fracture heals by endochondral ossification, with the formation of a cartilaginous callus around the fracture; large defects, such as 3.5 mm critical-sized defects do not heal completely and thus are used to model bone fracture non-union¹⁶. In this study, the plate fixation protocol of the axolotl femur prior to the osteotomy using the example of a 0.7 mm fracture gap was established with the ultimate goal of comparing axolotl bone healing to that of the mouse⁹.

After osteotomy, the fractures underwent the process of endochondral ossification, albeit slower than in mice, possibly due to the aquatic lifestyle of axolotls and slower cell division rates. In the method presented here, the 0.7 mm gap osteotomy with rigid plate fixation is shown; however, other gap sizes and semi-flexible fixators, as well as plates of different materials, are potentially possible. Overall, the method presented here can be used for standardized bone fixation and will be helpful for studies comparing axolotl limb regeneration to bone healing or studying bone healing in axolotls under different conditions to ensure standardized fracture fixation.

Protocol

The following procedure was performed with approval from the Magistrate of Vienna (GZ: MA 58-65248-2021-26). 5-8 years old, ≥ 20 cm snout-to-tail tip (snout to the tip of the tail) long axolotls (Ambystoma mexicanum) were used for fracture surgery and amputations. Both males and females were used for the surgeries. Axolotls were bred in the Research Institute of Molecular Pathology facility. Pain and risk of infections were managed with proper analgesics and antibiotics to ensure a successful outcome. The reagents and equipment used for the study are listed in the Table of Materials.

1. Animal preparation

1. Bath the animal in 0.03% benzocaine solution for about 15-20 min until full sedation is reached and there is no reflexive movement upon limb touching with tweezers.
2. Place the animal with the ventral side down on wet paper towels soaked in 0.03% benzocaine solution and cover it with benzocaine-soaked paper towels. The skin of aquatic animals, such as axolotls, is sensitive to drying, and thus, it is essential to cover the body surface to prevent dehydration and ensure cutaneous (skin) respiration.
3. Stretch out the hindlimb to be operated using ring forceps. Do not apply disinfection reagents, such as ethanol, since axolotl skin is sensitive to chemicals and easily irritated. Instead, use 0.7x PBS (A-PBS) with 50 U/mL penicillin and 20 µg/mL streptomycin for cleaning the limb and later on for bone irrigation upon sawing.
   NOTE: The infection is generally not a concern for the surgeries performed on axolotls. However, due to the aquatic nature of these animals and the sutures placed on the skin surface, we recommend the use of antibiotics to prevent any contamination of the surgical site.

2. Surgery

NOTE: Sterilize all surgical tools. Common sterilization methods such as heat sterilization, autoclaving, or washing in 70% ethanol, followed by thorough removal of the alcohol remnants, are suitable for this purpose. If operating on multiple animals, sterilize tools in between using a hot bead sterilizer or 70% ethanol.

1. Make a lateral longitudinal incision (1.5-2 cm) with a scalpel above the femur bone spanning the whole thigh in the upper hind limb. In order to do this, palpate the bone before cutting the skin.
2. Carefully displace the muscles and nerves from the surgery site without cutting. Use bowed forceps to do it efficiently.
3. Gently put bowed forceps under the femur to expose it for the surgery.
4. Place a rigid 7.75 mm 4-hole fixator plate along with the femur diaphysis, avoiding touching the joints, and secure it in the aligned position with forceps.
5. Use four 2 mm titanium screws to attach the bone to the plate.
   NOTE: The screws used in this protocol have a complex design and consist of 4 parts: the main part (will be screwed into the bone), screw head (allows removal of the screws and plate using the square box wrench), narrower neck (used as a breaking point once the screw is tightened in the bone) and a screw handle (used for attaching to the screwdriver and the saw-guiding device).
6. The order of screw attachment is important. Start with the inner screws first, and then the two outer screws to ensure the plate is aligned with the bone axis. Use a manual drill to create the first hole in the bone for easy insertion of the screw, followed by 1^st screw placing. Drill in the middle of the bone circumference to avoid thinner bone on one side, which may result in spontaneous bone fracture. Use irrigation with 0.7x PBS + 1% Pen/Strep during drilling. Do not break off the handle of the 1^st (optional: 1^st and 2^nd) screw(s).
7. Apply the saw-guiding device onto the 1^st (optional: 1^st and 2^nd ) screw(s) and align it with the bone and plate.
   NOTE: In this protocol, a plate, screws, a saw-guiding device, and a saw are provided by the same manufacturer and optimized to fit each other. The saw-guiding device can come in different sizes to be compatible with different plates and saw sizes.
8. Use the saw-guiding device to drill and insert the rest of the screws. Ensure alignment of the plate with the bone. Break off the handles of the screws.
9. Place a piece of plastic film (6-7 mm by 4-5 cm), sterilized by wiping with 70% ethanol, under the femur to prevent soft tissue damage during the osteotomy process.
   NOTE: For this purpose, a piece of plastic film, cut off a suture wrapping, is used. Alternatively, the plastic side of the bag for heat sterilization can be used.
10. Place Gigly wire saw between bone and protection film.
11. Cut bone using the 0.66 mm Gigly wire saw, creating a single 0.7 mm cut in the femur. Use constant irrigation with 0.7x PBS + 1% Pen/Strep during sawing to minimize tissue damage and friction.
12. Remove the saw, and saw guide and use a screwdriver to break off the screw handles from the screws.
13. Remove the protection film, and irrigate the surgery site with 0.7x PBS + 1% Pen/Strep.
14. Cover the top of the plate and screws with sterile bone wax to protect skin and muscles from irritation by the edges of the screws.
15. Place muscles and skin on top of the wax.
16. Close the incision site with a 7.0 synthetic (polypropylene/polyethylene) suture using simple interrupted stitches. Synthetic suture is used to minimize contamination with water-born bacteria and fungi.

3. Postoperative management

1. To reawaken the animal, place it in a tank with fresh artificial pond water, supplemented with 50 U/mL penicillin and 20 µg/mL streptomycin.
2. Observe the animal to start moving gills, making steps, and swimming - usually within 1 h post-surgery.
3. Keep the animal for 3 days in artificial pond water with 50 U/mL penicillin and 20 µg/mL streptomycin before returning it to the holding tank. To ensure analgesia, add Butorphanol (0.5 mg/L water).
4. Ensure that the sutures remain in place and proper wound healing occurs.

Representative Results

The surgical procedure described here (Figure 1) lasts between 20 min and 30 min and requires a surgeon and an assistant. Optionally, use a binocular dissection microscope or magnifying glass system.

Figure 1: Schematics of the surgical procedure and the experimental setup. (A) The femur is exposed by skin dissection and pulling muscles away from the surgery field. (B) A titanium plate is aligned with the femur. (C) 1^st inner hole is created using a manual drill. (D) A screw is placed into the 1^st hole. (E) The saw and drill guide is inserted and attached to the handle of the 1^st screw. (F) The rest of the screws are attached in the indicated order. (G) The saw and drill guide are carefully removed, and the handles of the screws are broken off while turning the screwdriver due to the higher stiffness of the bone in comparison to the screw neck. (H) Safety foil and Gigly saw are placed under the bone. (I) Osteotomy is performed under constant irrigation of the cutting site. (J) Screw heads are covered with sterile bee wax prior to replacing muscle and closing skin with stitches. (D,E,F): the numbers indicate the sequence in which holes are drilled and screws are placed. Please click here to view a larger version of this figure.

Special attention has to be paid to the plate-to-bone alignment and plate attachment steps (Figure 2). The variance in axolotl age leads to slight differences in femur size, shape, and ossification grade. Thus, in some cases, it was challenging to find a good plate position to ensure enough space for screw attachment. In total, fixation failure was observed in 23.5% of animals, which could be a result of narrower or less ossified bones in younger animals (Figure 3). Fixation failure resulted in a misalignment of the osteotomized bone. This was detectable by the presence of detached screws, displacement of the bone fragments with misalignment from the bone axis of >20°, or a pronounced lateral displacement. While the first could be observed when harvesting the samples, the others may not be obvious until microCT analysis is done. Whereas in properly aligned limbs, granulation tissue was located in the fracture gap 3 weeks post-surgery, in the misaligned samples, it appeared much larger, possibly in an attempt to stabilize the bone.

Figure 2: Overview of the surgical procedure and resulting animal with left hind limb osteotomy. (A) The main steps of the surgery are displayed: femur exposure, correct alignment of the titanium plate, using a saw guide for creating holes in the bone using a manual drill and application of the screws, using protective film and saw guide (yellow arrow) for osteotomy, 0.7 mm femur fracture (white arrow), covering the screw heads with bee wax (purple arrow) and the end result with closed limb. (B) Animal after surgery. The arrow indicates the left hind limb, where the osteotomy surgery was performed. Please click here to view a larger version of this figure.

Using bee wax prior to skin stitching (Figure 2) reduced the postoperative re-opening of the skin above the fixation plate. The limb remained functional and could be used for standing and walking shortly after anesthesia was removed.

Figure 3: Representative results of axolotl plate-fixed osteotomy. (A) MicroCT scans of intact and fractured femurs. Note a gap in the bone 3 weeks post-fracture. 23.5% of limbs displayed failed fixation, resulting in bone misalignment. In the fixated samples, hard callus is forming at 6-9 months post-fracture. (B) Safranin O/Light Green staining of intact and fractured femurs. Note a gap with granulation tissue in the bone 3 weeks post-fracture. At 6 months post-fracture, the hard callus consists of woven bone and cartilage. Scale bars: 1 mm. Please click here to view a larger version of this figure.

MicroCT and histological analysis (Safranin O/Light Green staining) allowed visualization of bone fragments' position and fracture gap size, as well as the bone healing state. MicroCT data was acquired with a nominal resolution of 10.05 µm in a commercially available specimen scanner and analyzed using compatible software (see Table of Materials) as described in Polikarpova et al.⁹. In contrast to mammals, where fracture of the same size (0.7 mm) is closed with cartilaginous and bony callus 3-5 weeks after surgery, no callus was observed 3 weeks post-surgery in axolotl. Typically, cartilaginous callus was visible at 3 months post-surgery⁹, and ossified bony callus - at 6 months post-surgery (Figure 3).

Discussion

The currently described method of femur plate fixation and osteotomy allows for its application in aquatic animals, such as Ambystoma mexicanum (axolotl). This surgical method was recently used to compare fracture healing and limb regeneration in axolotls to fracture healing in mice⁹. As in mice, a 4-hole fixator plate can be attached to the bone with self-breaking screws, and a Gigly saw can be used to create a fracture of uniform size¹⁵. Plate fixation facilitates gap size retention and ensures bone fragment alignment. The analgesia method was also optimized by implementing butorphanol bathing as it is less invasive (in comparison to buprenorphine injections), which is not reported to influence bone healing (as with carprofen).

Unlike in the method described for mouse¹⁶, a plastic film is used to protect underlying tissue prior to osteotomy. Also, due to the aquatic nature and less pronounced limb musculature, axolotls required a beeswax protection patch on top of the plate and screws, as well as special attention had to be paid to using synthetic skin stitches, as biofilms were collected on the absorbable sutures (polyglactin 910-based). The penicillin-streptomycin is utilized as part of postoperative treatment to prevent wound and suture contamination.

The method was optimized for 5-8-year-old, ≥20 cm snout-to-tail axolotls and thus limited to large, rather old animals. This could also be the reason for slower than mammalian bone healing due to overall lower cell proliferation rates in such old animals. However, this might be beneficial if one needs to resolve the finer steps of bone healing¹⁹. Another important feature of the method is the commercial nature of the main consumable materials used in the protocol: fixation plate, screws, and Gigly saw. Due to the optimized compatibility of these devices, bone defects of reliable size and distance from the bone ends are created. In the current protocol, the application of a titanium metal plate on axolotl bones is described. For mice, PEEK plastic plates were developed to ease microCT imaging of the samples¹⁷. The PEEK plates in axolotls were also tested with similar results; however, they could be applied only in larger animals due to the slightly higher volume of the plastic fixator plates in comparison to the metal plates.

In this study, we used 5-8-year-old animals and observed fixation failure in 23.5% of all surgeries, with higher failure rates in the younger animals. Thus, we recommend using older animals with a higher degree of bone ossification. The failure rate was independent of gender; thus, both sexes are equally qualified. For sample size planning, approximately 20% sample loss due to the unfavorable outcome should be calculated. The exact number of animals depends on the experimental design and the number of conditions to be analyzed; however, the recommended minimum animal number per group should be 5+20% = 6 animals.

Overall, this method gives a significant improvement over the previously used non-fixated or neighbor bone-fixated surgeries. Moreover, using this method, it is possible to study large axolotls that have bones similar in size and maturation to those used in mouse studies. This is important since microCT is only possible in large axolotls due to the degree of ossification.

The method is an important advancement in the field as it allows for direct comparison of axolotl limb regeneration to fracture healing, with controlled gap size and proper bone alignment (in 76.5% of the surgeries performed). Moreover, with PEEK plastic fixation plates, microCT, and X-rays can be used for bone healing process evaluation. Finally, the surgery described here can be adapted for other aquatic amphibians, such as newts and frogs, further facilitating cross-species studies of bone fracture healing.

Materials

Name	Company	Catalog Number	Comments
0.66 mm Gigly wire saw	RISystem	RIS.590.120
7.0 Optilene suture	Braun	C3090538
Benzocaine	Sigma-Aldrich	E1501	dilute to 0.03%  prior to using
Butorphanol (Butomidor 10 mg/mL)	Richter Pharma AG	-	dilute to 0.5 mg/L  prior to using
Drill bit 0.30 mm	RISystem	RIS.590.200
Dumont #5 Forceps - Standard/Inox	Fine Science Tools	11251-20
Hand drill	RISystem	RIS.390.130	better to have at least 3 pieces
Micro CT data analyzer	Bruker, Billerica, MA, USA	SkyScan NRecon software
Micro CT specimen scanner	Bruker, Billerica, MA, USA	SkyScan 1172
Moria MC31b Iris forceps - smooth, curved, 10 cm	Fine Science Tools	11373-12FST	2 pieces
MouseFix Drill-&Saw guide 1.75 mm, rigid	RISystem	RIS.301.102
MouseFix plate 4 hole, rigid	RISystem	RIS.401.110
MouseFix screw, L =2.00 mm	RISystem	RIS.401.100	need 4 per bone
Narrow Pattern Forceps	VWR	FSCI11002-12
penicillin/streptomycin	Gibco	15140-122
Ring forceps	Fine Science Tools	11103-09
scalpel #15	B Braun, Thermo Fischer Scientific	5518032
Square box wrench 0.50 mm	RISystem	RIS.590.111
Sterile bone wax, 2.5 g	Ethicon, Johnson & Johnson	W810
Student Fine Scissors - Straight/11.5cm	Fine Science Tools	91460-11